The Effect of Water Activity on the Growth and Respiration of Pseudomonas fluorescens

The growth rate of Pseudomonas fluorescens was greater and continued at lower water activity ( a _w) values when glycerol controlled the a _w of glucose minimal medium than when the a _w was controlled by NaCl and sucrose. Growth was not observed below 0·945, 0·970 and 0·964 a _w when glycerol, sucrose and NaCl respectively controlled the a _w. The catabolism of glucose, Na lactate and DL-arginine as measured by respirometry was completely inhibited at a _w values greater than the minimum for growth when the a _w was controlled with NaCl. When the a _w was controlled with glycerol, catabolism of the three substrates continued at a _w values significantly below the a _w for growth on glucose. Catabolism of glucose in the presence of sucrose occurred at a level below the minimum growth a _w but catabolism of the other two substrates ceased at a _w values greater than the minimum growth a _w. Arrhenius plots between 10° and 34°C of the growth rate in glucose minimal medium at 0·98 a _w showed that the order of inhibition was sucrose > NaCl > glycerol. The order of inhibition differed when Arrhenius plots of catabolism of glucose was examined between 10° and 34 °C, namely NaCl > sucrose > glycerol. The mechanism of action of solutes controlling a _w is discussed.     

Water Relations of Glucose-catabolizing Enzymes in Pseudomonas fluorescens

Examination of the catabolism of glucose via the Entner-Doudoroff pathway by standard enzyme assays showed that the activity of glucose-6-phosphate dehydrogenase, glucokinase and 2-ketoglu-conokinase plus phosphoketogluconate reductase was completely inhibited at a _w values less than 0.965, 0.98 and 0.96 respectively when NaCl was used to adjust the a _w. The other glucose-catabolizing enzymes were inhibited to a lesser degree. When sucrose was used to control a _w, glucokinase and glucose-6-phosphate dehydrogenase were inhibited at 0.92 a _w but the other enzymes remained active below 0.86 a _w. Enzymes were relatively active at reduced a _w when adjusted with glycerol and most remained active even at 0.80 a _w. When a _w was controlled by potassium glutamate, the activity of glucokinase and glucose-6-phosphate dehydrogenase was markedly less inhibited than by NaCl at similar a _w. Possible reasons for the variation in activity by glucose-catabolizing enzymes in response to a _w controlled by various solutes could be location of the enzyme in the cell, ability of the solute to penetrate the cell and ability to withstand high salt and sucrose concentrations. When the a _w of the growth medium was reduced to 0.98 by glycerol, NaCl and polyethylene glycol 400, levels of glucokinase were significantly reduced while higher levels of glucose dehydrogenase and gluconate dehydrogenase were induced. This suggests that reduction in a _w could regulate the routes of catabolism in the Entner-Doudoroff pathway. When sucrose was used to control a _w of the growth medium high levels of most enzymes were induced, suggesting catabolism of the sucrose by the organism.     

Radiation Resistance of Spores of Bacillus subtilis and B. stearothermophilus at Various Water Activities

S ummary . The radiation resistance of spores of Bacillus subtilis and B. stearothermophilus was determined under anoxic conditions in water vapour and in aqueous solutions of glucose and glycerol at various water activities. In water vapour, resistance of both kinds of spores increased slightly with decreasing water activity ( a _w). In glycerol solutions, a phase of rapid increase in resistance with decreasing a _w was followed by a slower increase on further reduction of a _w. This second phase was almost parallel to the water vapour curve, which, however, showed considerably lower resistance. The initial phase of rapid increase in resistance was also observed in glucose solutions.     

Microbial growth at reduced water activities: studies of aw prediction in solutions of compatible solutes

Studies were made of the prediction of intracellular water activity ( a _w) of microorganisms grown at reduced a _w. These included the determination and prediction of a _w of potassium L-glutamate solutions and the prediction of a _w in mixed solutions of various compatible solutes and macromolecules. Compatible solutes studied were L-proline, glycerol, potassium chloride and D-arabitol. The results indicate that a _w predictions of such mixtures may be done with acceptable accuracy by neglecting solute-solute interactions.     

Chrysosporium species, potential spoilage organisms of chocolate

Standard methods of analysing foods for the presence of moulds are inadequate for thedetection of genera such as Chrysosporium which do not grow at the high wateractivities of most mycological media. The use of malt, yeast, 50% glucose agar (MY50G) insealed containers as an enrichment medium allowed time for germination and growth ofheat-stressed spores. Three Chrysosporium spp., C. xerophilum Pitt, C. inops (Carmichael) and C. farinicola (Burnside) Skou, were isolated fromcommercial chocolate bars with a water activity (a _w ) of approximately0·28. Chrysosporium inops was isolated from commercial milk crumb and anew Chrysosporium sp. was isolated from Ghanaian cocoa beans. In chocolates madeby coating MY50G agar (a_w = 0·89) with chocolate (a_w = 0·27) containing C. inops arthroconidia, two types of deterioration were seenafter storage. The first was fat bloom due to recrystallization of the cocoa butter on the outer andinner chocolate surface. The second was growth of C. inops which occurred on theinside chocolate surface adjacent to the MY50G agar filling and on the outside surface afterholding at 92% equilibrium relative humidity (erh) for 12 d. There was some evidence that C. inops could grow on the outside of chocolates held at 5·7% erh after 4months' storage at 25 °C. The appearance of the white fungal growth was not unlikefat bloom to the naked eye but was clearly different with the electron microscope.     

Chemostat adaptation of Escherichia coli B/r/1 to low water activity

Chemostat cultures of Escherichia coli B/r/1 under conditions of glucose limitation in a salts medium at high water activity ( a _w of 0.999) and at an a _w of 0.987, controlled by NaCl, have been compared. The system was run at dilution rates above 0.035 h^-1. The results suggested that the organism adapted to the lower a _w of the medium as no significant change was observed in the energy requirement for maintenance, although the maximum molar growth yield for glucose decreased by 23.2%. Such cultures showed also a shorter lag and a higher growth rate in batch at an a _w of 0.987, as compared with cultures initiated with an unadapted ( a _w of 0.999) inoculum.     

Microbial growth at reduced water activities: some physicochemical properties of compatible solutes

C hirife , J., F avetto , G. & F erro F ontán, c. 1984. Microbial growth at reduced water activities: some physicochemical properties of compatible solutes. Journal of Applied Bacteriology 56 , 259–268.
The water activities ( a _w) of solutions of D-arabitol (a major compatible solute in yeasts grown at reduced a _w) and also of related polyols (L-arabitol, xylitol and ribitol) were determined at 25C. The a _w of saturated solutions of L-proline and monopotassium L-glutamate (solutes which accumulate in bacterial cells at reduced a _w) were also determined. The viscosities of L-proline, monopotassium L-glutamate and D-arabitol solutions were measured at 25, 30, 35 and 40C for various concentrations up to high molalities. The relative viscosity curves were described using a theoretical model for the concentration dependence of viscosity. No relationship of viscosity of the electrolytes to effectiveness as 'compatible solutes' were evident, neither were the compatible solutes particularly effective 'structure makers' or 'structure breakers.'     

Determination of optimal growth parameters for the bioincising fungus Physisporinus vitreus by means of response surface methodology

Aim:  To evaluate the influence of water activity ( a _w), temperature and pH on the radial growth and lag phase of Physisporinus vitreus (E-642), a basidiomycete was used in the biotechnological process of bioincising.
Methods and Results:  Radial growth was monitored for 20 days on malt extract agar medium. Five levels of a _w (0·998, 0·982, 0·955, 0·928, 0·892) were combined with three incubation temperatures (10, 15, 20°C) and three pH values (4, 5, 6). Data analyses showed a highly significant effect of a _w and temperature ( P <  0·0001) and a significant effect of pH ( P <  0·05). The radial growth rate and lag phase of P. vitreus were very sensitive to a _w reduction. Although P. vitreus was able to grow at all the selected temperatures and pH values, the lag phase increased with decreasing a _w and growth became inhibited at a _w = 0·955. Optimal conditions for growth of P. vitreus were a _w = 0·998, 20°C and pH 5. The response surface model provided reliable estimates of these growth parameters and confirmed a greater dependence on a _w than on temperature or pH under in vitro conditions.
Conclusions:  Low levels of a _w can prevent growth of P. vitreus , so wood moisture content should be adjusted accordingly.
Significance and Impact of the Study:  Implementation of these results should contribute towards the optimization and efficiency of bioincising.     

The Effect of Water Activity and the aw Controlling Solute on Spore Germination of Bacillus stearothermophilus

The germination of spores of Bacillus stearothermophilus was studied in nutrient broth in relation to the water activity ( a _w) of the medium, the nature of the a _w controlling solutes glycerol, sucrose, KCl, and NaCl, and temperature. Quantitation of germination was based on the change of the phase-bright spore to phase-dark. Activation of spores was by exposure to 100°C/10 min in a medium of the same composition as that used for germination.
Of the four solutes used, sucrose proved most inhibitory to germination, especially in the upper part of the temperature range 38-75°C, glycerol was the most favourable whereas KCl and NaCl, whose effect was almost identical, occupied an intermediate place. The glycerol effect became more pronounced as the a _w of the medium decreased towards 0.960, becoming inhibitory thereafter.
The solute effect on spore germination followed a pattern that related to the class of solute, i.e. electrolyte or non-electrolyte, and its cell penetration characteristics.
Solute penetration during heat activation and germination was considered as the major germination factor and was associated with the osmoregulation mechanism within the spore proposed recently as the basis of spore dormancy and resistance.     

Spirochaeta smaragdinae sp. nov., a new mesophilic strictly anaerobic spirochete from an oil field

An obligately anaerobic spirochete designated strain SEBR 4228^T (T = type strain) was isolated from an oil field of Congo, Central Africa. The strain grew optimally with a sodium chloride concentration of 5% (sodium chloride concentration growth range 1.0–10%) at 37°C (growth temperature range 20–40°C) and pH of 7.0–7.2 (pH growth range pH 5.5–8.0). Strain SEBR 4228^T grew on carbohydrates (glucose, fructose, ribose, d -xylose, galactose, mannitol and mannose), glycerol, fumarate, peptides and yeast extract. Yeast extract was required for growth and could not be replaced by vitamins. It reduced thiosulfate and sulfur, to H₂S. Glucose was oxidised to lactate, acetate, CO₂ and H₂S in the presence of thiosulfate but in its absence lactate, ethanol, CO₂ and H₂ were produced. Fumarate was fermented to acetate and succinate. The G+C content of strain SEBR 4228^T was 50%. Strain SEBR 4228^T was spiral shaped measuring 5–30 by 0.3–0.5 μm and was motile with a corkscrew-like motion. Electron microscopy revealed the presence of periplasmic flagella in a 1-2-1 arrangement. Strain SEBR 4228^T possessed features typical of the members of the genus Spirochaeta . 16S rRNA sequence analysis revealed that it was closely related to Spirochaeta bajacaliforniensis (similarity 98.6%). The lack of DNA homology with S. bajacaliforniensis (38%), together with other phenotypic differences, indicated that strain SEBR 4228^T is a new species, which we have designated Spirochaeta smaragdinae . The type strain is SEBR 4228^T (= DSM 11293).     

Production and storage of Rhizobium leguminosarum cell concentrates for use as inoculants

Recovery of Rhizobium leguminosarum cells by centrifugation after growth in an industrial fermenter was 100-fold higher when cells were grown on yeast extract (5 g/1) as sole source of carbon and nitrogen when compared with the yields recovered when cells were grown in standard mannitol-yeast extract medium. Methods of storing concentrated suspensions of R. leguminosarum were investigated. Freeze-drying caused a marked decrease in viable cell numbers. Viable cell numbers of bacterial concentrates stored in peat decreased steadily from 10₁₁-10₁₂ cfu/g to 10₉ cfu/g or less during 26 weeks storage at room temperature or at 4°C. Cell concentrates stored in 40% glycerol at — 20°C maintained viable numbers higher than 10₁₁ cfu/ml during a 76 week storage period.     

Effect of water activity and temperature on growth and fumonisin B1 and B2 production by Fusarium proliferatum and F. moniliforme on maize grain

S. MARIN, V. SANCHIS, I. VINAS, R. CANELA AND N. MAGAN. 1995. The effect of different water activities ( a _w, 0.968, 0.956, 0.944, 0.925) and temperature (25°C and 30°C) on colonization and production of fumonisin B₁ (FB₁) and B₂ (FB₂) on sterile layers of maize by Fusarium proliferatum and F. moniliforme isolates was determined over periods of 6 weeks. Generally, both F. moniliforme and F. proliferatum grew faster with increasing a _w and best at 30°C. All three isolates produced more FB 1 than FB₂ regardless of a _w or temperature. Very little FB₁ and FB₂ were produced at 0.925 a _w, with maximum produced at 0.956 and 0.968 a _w at both temperatures tested. Most FB₁ and FB₂ were produced by F. moniliforme (25N), followed by F. proliferatum isolates (73N and 131N). At all a _w levels and both temperatures there was an increase in FB₁ and FB₂ concentration with time. Statistical analyses of a _w, temperature, time, two- and three-way interactions showed some significant differences between isolates and FB₁ and FB₂ production.     

The survival of Salmonella enterica serovar Typhimurium DT104 and total viable counts on beef surfaces at different relative humidities and temperatures

Aims:  The aim of this study was to investigate changes in Salmonella and total viable count (TVC) survival on beef carcass surfaces stored for 72 h under different combinations of relative humidity (i.e. RH 75% or 96%) and temperature (5°C or 10°C).
Methods and Results:  The influence of low water activity ( a _w) and temperature on the survival and growth of Salmonella enterica serovar Typhimurium DT104 and the aerobic mesophilic flora on meat pieces from different sites on beef carcasses was investigated, under controlled conditions (75% or 96% RH; 5 or 10°C) in an environmental cabinet. Salmonella counts declined during storage at low a _w (75% RH) conditions at 5°C or 10°C. Salmonella counts increased during storage at high a _w (96% RH) at 10°C only. At 5°C, TVCs increased during storage at high a _w, but not at low a _w. TVCs increased on all samples from carcasses stored at high or low a _w at 10°C, except those samples taken from areas of surface fat.
Conclusions:  This suggests that substrate composition dictates growth rates under low a _w conditions. The results are discussed in terms of the possible protective effects of substrate osmolyte accumulation in bacterial survival and/or growth.
Significance and Impact of the Study:  The data obtained in this study provides useful insights on the influence of a _w and temperature on pathogen survival on meat surfaces at chill temperature.     

Growth of Fusarium moniliforme and its biosynthesis of fumonisin B1 on maize grain as a function of different water activities

The production of fumonisin by Fusarium moniliforme during its growth on maize depends on extrinsic factors. In particular, experiments on maize grain at different water activities ( a _w)(1, 0.95, 0.90, 0.85) have demonstrated the influence of a _w on fumonisin biosynthesis, and on fungal growth defined by measurement of ergosterol levels. Fumonisin levels dropped threefold when a _w was lowered by 5%, but growth rate was unchanged. A 10% reduction in a _w from 1 to 0.90 resulted in a 20-fold drop in fungal growth, and fumonisin production was reduced 300-fold. At a threshold a _w of 0.85–0.86, F. moniliforme exhibited virtually no measurable metabolic activity, and hence no fumonisin production.     

Effect of temperature and water activity on in vitro germination of Monilinia spp.

Aims:  This study evaluated the effect of temperature (0–38°C) and water activity ( a _w: 0·87–0·99) on the lag phase prior to germination and the percentage of germination over time for Monilinia laxa , Monilinia fructicola and Monilinia fructigena .
Methods and Results:  More than 80% of viable conidia germinated at 25°C and 0·99 a _w within 2 h for M. fructicola and M. fructigena and 4 h for M. laxa . There was no germination at 38°C, and all three Monilinia spp. germinated at 0°C. At the lowest a _w (0·87), none of the Monilinia spp. was able to germinate at any of the incubation temperatures studied. Whereas at 0·90 a _w, conidia were only able to germinate at 15, 25 and 30°C for the three species studied, except for M. fructicola at 15°C. In contrast, at 0·95, 0·97 and 0·99 a _w, germination occurred at all studied temperatures less 38°C. Generally, the lag phase was longer at low levels of a _w (0·90–095), and differences were more evident as temperatures were far from the optimum (0–5°C).
Conclusions:  Germination and lag phase period were markedly influenced by temperature and a _w, and in general when conditions of temperature and a _w were suboptimal, the lag phase was longer and the percentage of germination was lower.
Significance and Impact of the Study:  Knowledge of the germination requirements of this fungus is important in order to understand their behaviour in natural situations and to provide baseline data required for the construction of new prediction models. Our study might be used to develop a predictive model to understand and control the disease caused by Monilinia spp.     

Water stress induced changes in concentrations of proline and total soluble sugars in nodulated alfalfa (Medicago sativd) plants

Susceptibility of alfalfa ( Medicago saliva L. cv. Aragón) nodules and leaves to water stress has been investigated. Nodule acetylene reduction activity (ARA), leaf CO₂ exchange rate (CER) as well as soluble protein, proline and total soluble sugar (TSS) contents were determined during drought. Water status was estimated as water potential (Ψ_w) and Relative water content (RWC) of the respective tissues. Maximum rates of ARA required higher Ψ_w than CER. Nodules had lower RWC for a given Ψ_w than leaves. Water stress reduced soluble protein content in both tissues; however, the decline in soluble protein content was detected at greater Ψ_w in nodules than in leaves. Proline and TSS increased in leaves and nodules, and again the threshold Ψ_w triggering such accumulation was higher in nodule tissues. Oior results suggest that alfalfa nodules are more susceptible to water shortage than leaves. Effects of accumulated TSS and proline upon leaf and nodule physiology are discussed in relation to protein stability (proline), pH control (proline) and osmotic adjustment (proiine and TSS). The TSS accumulation induced by water stress suggests that substrate shortage would not be the primary effect of drought on nodule activity.     

The Effect of Sugars and Polyols on the Heat Resistance of Salmonellae

S ummary . The heat resistance at 65° of 3 strains of salmonellae in solutions of sugars or polyols was enhanced as the concentration of the solutes increased. There was no linear relationship between heat resistance and water activity ( a_w ), but for all solutes except glycerol there was a linear relationship between log D ₆₅ and concentration (% w/w) of solute. Comparison of D ₆₅ at a particular a_w or percentage (w/w) solute concentration showed that the value decreased in the order: sucrose > glucose > sorbitol > fructose > glycerol. In glycerol, D ₆₅ values were always very much lower than in any other solute. With sucrose–glycerol or sucrose–glucose mixtures, heat resistance depended both on the total concentration (% w/w) of solutes present and also on the a_w of the solution.     

Modelling the growth of Weissella cibaria as a function of fermentation conditions

Aims:  To investigate the effect of pH, water activity ( a _w) and temperature on the growth of Weissella cibaria DBPZ1006, a lactic acid bacterium isolated from sourdoughs.
Methods and Results:  The kinetics of growth of W. cibaria DBPZ1006 was investigated during batch fermentations as a function of pH (4·0–8·0), a _w (0·935–0·994) and temperature (10–45°C) in a rich medium. The growth curve parameters (lag time, growth rate and asymptote) were estimated using the dynamic model of Baranyi and Roberts (1994. A dynamic approach to predicting bacterial growth in food. Int J Food Microbiol 23, 277–294). The effect of pH, a _w and temperature on maximum specific growth rate (μ_max) were estimated by fitting a cardinal model. μ_max under optimal conditions (pH = 6·6, a _w = 0·994, T  = 36·3°C) was estimated to be 0·93 h⁻¹. Minimum and maximum estimated pH and temperature for growth were 3·6 and 8·15, and 9·0°C and 47·8°C, respectively, while minimum a _w was 0·918 (equivalent to 12·2% w/v NaCl).
Conclusions:  Weissella cibaria DBPZ1006 is a fast-growing heterofermentative strain, which could be used in a mixed starter culture for making bread.
Significance and Impact of the Study:  This is the first study reporting the modelling of the growth of W. cibaria , a species that is increasingly being used as a starter in sourdough and vegetable fermentations.     

Relations between carbohydrate, water status and adventitious root formation in leafy pea cuttings rooted under various levels of atmospheric CO2 and relative humidity

Three levels of atmospheric CO₂ and 2 levels of relative humidity (RH) during the rooting period were tested for their effect on several factors presumed to influence adventitious root formation in leafy pea ( Pisum sativum L. cv. Alaska) cuttings. Compared to normal CO₂ levels (350 μl l⁻¹), neither 1800 nor 675 μl l⁻¹ CO₂ affected the rooting percentage or the number of roots per cutting. However, 1800 μl l⁻¹ CO₂ increased root and shoot dry weight, root length, carbohydrate levels in the base of the cuttings and water potential (Ψ_w) of cuttings compared to normal levels of CO₂. Compared to 87% RH. 55% RH decreased all of the above parameters, including the number of roots per cutting. A polyvinyl chloride antitranspirant (which partially blocks stomata and slows photosynthesis) applied simultaneously with 87% RH increased Ψ_w and root length but lowered all of the other above parameters, compared to 87% RH without antitranspirant. Increasing current photosynthate (products of photosynthetic activity after excision), carbohydrate, or Ψ_w either alone or together was associated with increased root system size but not necessarily with increased rooting percentage or root number. The data are consistent with a hypothesis that the number of roots per cutting increased with increasing current photosynthate and carbohydrate until some other factor became limiting. Also, the effect of Ψ_w on rooting percentage and root number was mediated through its effect on current photosynthate and carbohydrate.     

Use of wheat bran as a nutritive supplement for the production of ethanol by Zymomonas mobilis

A strain of Zymomonas mobilis (ZYM-TS 1) was isolated from fermenting palm wine (toddy). In addition to glucose, sucrose, and fructose, the organism utilized hydrolysates of corn ( Zea mays ) flour, corn starch and ragi ( Eleusine coracana ) flour. Amounts of ethanol produced in media (adjusted to 10% (w/v) total carbohydrates) fortified with wheat bran extract only, were comparable with those obtained from the defined media containing yeast extract, (NH₄)₂SO₄, KH₂PO₄, and MgSO₄.7H₂O. The results indicate that wheat bran extract can supply all the necessary nutrients required for ethanol production by Zymomonas mobilis .